Radiosynthesis, structural identification and in vitro tissue binding study of [18F]FNA-S-ACooP, a novel radiopeptide for targeted PET imaging of fatty acid binding protein 3

Background Fatty acid binding protein 3 (FABP3) is a target with clinical relevance and the peptide ligand ACooP has been identified for FABP3 targeting. ACooP is a linear decapeptide containing a free amino and thiol group, which provides opportunities for conjugation. This work is to develop methods for radiolabeling of ACooP with fluorine-18 (18F) for positron emission tomography (PET) applications, and evaluate the binding of the radiolabeled ACooP in human tumor tissue sections with high FABP3 expression. Results The prosthetic compound 6-[18F]fluoronicotinic acid 4-nitrophenyl ester was conveniently prepared with an on-resin 18F-fluorination in 29.9% radiochemical yield and 96.6% radiochemical purity. Interestingly, 6-[18F]fluoronicotinic acid 4-nitrophenyl ester conjugated to ACooP exclusively by S-acylation instead of the expected N-acylation, and the chemical identity of the product [18F]FNA-S-ACooP was confirmed. In the in vitro binding experiments, [18F]FNA-S-ACooP exhibited heterogeneous and high focal binding in malignant tissue sections, where we also observed abundant FABP3 positivity by immunofluorescence staining. Blocking study further confirmed the [18F]FNA-S-ACooP binding specificity. Conclusions FABP3 targeted ACooP peptide was successfully radiolabeled by S-acylation using 6-[18F]fluoronicotinic acid 4-nitrophenyl ester as the prosthetic compound. The tissue binding and blocking studies together with anti-FABP3 immunostaining confirmed [18F]FNA-S-ACooP binding specificity. Further preclinical studies of [18F]FNA-S-ACooP are warranted. Supplementary Information The online version contains supplementary material available at 10.1186/s41181-024-00245-3.

six are peptides or peptide analogues (http:// www.radio pharm aceut icals.info/ radio pharm aceut icals.html).In the international research community, much research endeavors have been devoted to the development of novel peptide-based radiopharmaceuticals and methodologies for peptide radiolabeling (Krecisz et al. 2021).We have been developing several new peptides and their radiolabeling methods for PET imaging of inflammation and cancer, targeting different types of biological targets (Käkelä et al. 2018;Li et al. 2013).Recently, using an in vivo phage display technique, Laakkonen and co-workers identified a brain tumor-homing peptide (named ACooP, sequence H-ACGLSGLGVA-NH 2 ) that targets the fatty acid binding protein 3 (FABP3, also known as mammaryderived growth inhibitor or heart-type fatty acid-binding protein) (Hyvönen et al. 2014).The targeting affinity and specificity of ACooP and its several variants have been studied in detail in different experimental settings (Feng et al 2015;Lico et al. 2021).FABP3 plays a significant role in cancer and neurodegenerative diseases (Kawahata et al. 2019;McKillop et al. 2019).For example, in a study involving 1331 patients with breast cancer, 94.3% of tumor samples were FABP3-positive, and this was associated with better 10-year survival rate (Nevo et al. 2010).As another example, FABP3 expression was colocalized with α-synuclein aggregates in the human brain tissue sections with synucleinopathies (Oizumi et al. 2021).Furthermore, FABP3 expression has been linked to several other diseases, as detailed in a recent review (Mallick et al. 2021).Given the growing body of clinical evidence implicating FABP3 expression in human diseases, we hypothesize that FABP3 could be a potential target for PET imaging.Accordingly, we set out to radiolabel ACooP peptide with fluorine-18 ( 18 F) for FABP3-targeted PET imaging applications.
In this work, we chose to use [ 18 F]1 as the prosthetic compound for radiolabeling of ACooP peptide (Fig. 2), as its precursor compound N,N,N-trimethyl-5-((4-nitrophenoxy)carbonyl)pyridine-2-aminium triflate (4) is commercially available and [ 18 F]1 is not Fig. 1 Examples of 18 F-prosthetic groups for N-acylation of biomolecules volatile, which is important from radiation safety point of view.Here, we demonstrate that it is feasible to use on-resin 18 F-fluorination to prepare [ 18 F]1, and [ 18 F]1 has high chemoselectivity towards S-acylation instead of the expected N-acylation in the case of ACooP peptide conjugation.Furthermore, we carried out structural characterization in details to confirm the chemical identity of the radiolabeled product [ 18 F]FNA-S-ACooP (Figs. 3, 4) and studied its in vitro binding using brain metastasis tissue sections from a patient with lung cancer.

Structural characterization of FNA-N-ACooP
To facilitate the structure identification of FNA-S-ACooP, reference compound FNA-N-ACooP was purchased as a custom synthesis and characterized with MS, 1D and 2D NMR (Additional file 1: Fig. S9-15).1D NMR analysis data were described as follow.δ 1 H(600 MHz;
Radiochemical purity and identity of [ 18 F]FNA-S-ACooP were analyzed by HPLC.Accordingly, a mixture of [ 18 F]FNA-S-ACooP (0.5-1.0 MBq) and 10 nmol of cold reference FNA-S-ACooP in 50 μL of PBS containing ascorbic acid (14 mM) was injected into a reversed-phase C18 column (Jupiter Proteo, 250 × 4.6 mm, 5 µm, 90 Å; Phenomenex) at a flow rate of 1 mL/min under radioactivity detection and ultraviolet (UV, wavelength 220 nm) detection.Solvent A was 0.1% TFA in water and solvent B was 0.1% TFA in acetonitrile.The HPLC elution gradient was from 27 to 42% B during 0-10 min.The decay-corrected radiochemical yield was 29.9% ± 2.3 (n = 4) and radiochemical purity was 96.6% ± 2.3.The molar activity was 36.2 ± 22.0 MBq/nmol at the end of synthesis.The total synthesis time was 181.5 ± 6.9 min from the end of bombardment.The shelf-life of [ 18 F]FNA-S-ACooP in the injectable formulation (PBS with 14 mM ascorbic acid) was investigated by taking samples at different time points up to 4 h for HPLC analysis as described above.

LogD 7.4 measurements
The distribution coefficient LogD 7.4 was measured by topping up 5 kBq of the tracer [ 18 F]FNA-S-ACooP with PBS (pH 7.4) to 600 µL and adding 600 µL of 1-octanol.The solution was mixed thoroughly for 3 min and then centrifuged for 3 min at 12,000 × g to separate the layers.Aliquots of 400 µL each were taken from both layers and the radioactivity was measured using a gamma counter.The tests were performed in triplicate.LogD 7.4 of [ 18 F]FNA-S-ACooP was calculated as follows with radioactivity decay correction: LogD = log 10 counts in the octanol phase counts in the PBS phase .LogD 7.4 of [ 18 F]FNA-S-ACooP was − 0.55 ± 0.01 (n = 3).

In vitro tissue binding and autoradiography
Cryosections (20 µm thickness, n = 4) of a brain metastasis sample from a patient with lung cancer were defrosted at 4 °C for 15 min followed by r.t. for 15 min and incubated in PBS (pH 7.4) at r.t. for 15 min.The slides were then incubated in PBS containing [ 18 F]FNA-S-ACooP at a radioactivity concentration of 0.018 MBq/mL for 45 min at r.t., rinsed twice with 4 °C PBS for 2 min, and dipped once with water at 4 °C.Slides were dried with gentle air flow and exposed to a BAS-TR2025 phosphor imaging plate (Fujifilm, Tokyo, Japan).After an exposure time of 18 h, the imaging plates were scanned with a BAS-5000 scanner (Fujifilm, Tokyo, Japan), and the autoradiography images were viewed with Carimas 2.10 software (Turku PET Centre, Turku, Finland, www.turku petce ntre.fi/ carim as/).The blocking experiments were performed with similar protocols as described above, except that the tissue cryosections were first incubated with molar excess of ACooP (5 μM) for 15 min before addition of [ 18 F]FNA-S-ACooP (0.5 nM).The radioactivity binding in the tissue samples was quantified as photostimulated luminescence units per square millimeter (PSL/mm 2 ) with the background radioactivity corrected.The collection of human tissue samples was approved by the ethical committee of Helsinki University Hospital, Finland.

Stability of [ 18 F]FNA-S-ACooP in rat plasma in vitro
The stability of [ 18 F]FNA-S-ACooP was assessed in vitro using plasma from a Sprague Dawley rat.After incubating [ 18 F]FNA-S-ACooP (0.2 MBq) in plasma (1.0 mL) at 37 °C for 5 min, 10 min, and 15 min, respectively, 50 μL samples were taken and plasma proteins precipitated with 50 µL acetonitrile.The mixture was then centrifuged, and the supernatant was injected into a reversed-phase C18 column for HPLC analysis at a flow rate of 5 mL/min, under radioactivity detection and UV detection.The HPLC solvent A was 0.1% TFA in water and solvent B was 0.1%TFA in acetonitrile.The elution gradient was from 10 to 50% B during 0-15 min.The experiments were performed in triplicates at each time point.The presence of intact [ 18 F]FNA-S-ACooP in the plasma samples was confirmed with the reference standard of [ 18 F] FNA-S-ACooP, and the presence of [ 18 F]FNA as one of the radiometabolites was confirmed with the reference standard of [ 18 F]FNA.[ 18 F]FNA was prepared by hydrolyzing [ 18 F]1 with 1 M NaOH followed by neutralization with 1 M HCl and phosphate buffer (0.1 M) to pH 6-7.

Preparation of 1 and FNA-S-ACooP
1 was prepared by an esterification reaction in the presence of EDC as the coupling agent and a catalytic quantity of DMAP.Upon acid work up and flash column purification, the product was obtained in solid form in 49% yield.The chemical structure of 1 was confirmed by NMR (Additional file 1: Fig. S1) and HRMS.FNA-S-ACooP was synthesized by using 1 as the acylation agent in the presence of ACooP peptide in a one-pot reaction at r.t.To isolate the product, a semi-preparative HPLC was performed and the factions were collected.After removing the solvents, FNA-S-ACooP was obtained as a solid in 89% yield.

Structural characterization of FNA-S-ACooP in comparison with FNA-N-ACooP
The chemical structure of FNA-S-ACooP was characterized by LC-MS and NMR analyzes, and the corresponding N-acylated compound FNA-N-ACooP was used as a reference.In the LC-MS/MS analysis (Additional file 1: Fig. S2), FNA-S-ACooP m/z [M + H] 1+ was 969.4612 (theoretical value 969.4622) and the retention time was 11.05 min.To assign the MS fragments, the raw data were searched against the peptide sequence, including the modifications, using Proteome Discoverer 3.0 connected to a Mascot search engine.The Mascot search engine found the FNA moiety attached to the sulfhydryl group at the cysteine side chain instead of amino group at alanine-1 (Ala1).The fragmentations that proved the attachment site were m/z 397.13 (CGL y and a fragmentations), 484.17 (CGLS y and b fragmentations), 513.19 and 541.19 (CGLSG y and a/b fragmentations) and 626.28 and 654.27 (CGLSGL y and a/b fragmentations).In the NMR analysis, the individual amino acid residues were recognized mainly from the 2D total correlation spectroscopy (TOCSY) and heteronuclear single quantum coherence (HSQC) spectra, and further confirmed with 2D nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear multiple bond correlation (HMBC) spectra (Additional file 1: Fig. S3-6).HMBC revealed the sequence based on the coupling of backbone amide protons with the adjacent carbonyl carbon.1D 1 H and 13 C measurements were used for exact chemical shift assignments (Fig. 4, and Additional file 1: Fig. S7-S8).Cysteine protons were located at (in ppm) 3.37 and 3.51 (Cysβ), 4.65 (Cysα) and 8.86 (CysNH).Sulfhydryl proton was not found in the spectra.Alanine-1 proton peaks were broadened except Ala1β (in ppm): 1.34 (Ala1β), 3.89 (1 H, br, Ala1α)) and 8.05 (3 H, br, Ala1NH 3 + ).Connections between Ala1 protons and FNA carbonyl carbon was missing but instead Cysβ protons were coupled to it on the HMBC spectrum.FNA carbonyl carbon was quite downfield at 188.07, which fits well to a thioester.
Next, we performed structural analyzes of custom-synthesized N-acylated compound FNA-N-ACooP by LC-MS and NMR (Fig. 4 and Additional file 1: Fig. S9-S15) as comparison with FNA-S-ACooP, and we indeed observed clear differences.FNA-N-ACooP m/z [M + H] +1 was 969.4617 which was identical as FNA-S-ACooP, but retention time was 13.03 min in comparison to that of FNA-S-ACooP at 11.05 min.The fragment ion spectra of m/z 969.46 looked different between the two samples.The spectrum of FNA-N-ACooP was simpler, and there were differences in fragment ions of the compounds.The Mascot search engine found the FNA moiety attached to Ala1 in FNA-N-ACooP.The fragmentations that proved the attachment site were m/z 246.13 (CGL y and a fragmentations), 588.28 (CGLSGLG y and b fragmentations) and 687.35 (CGLSGLGV y and b fragmentations).Compared to FNA-S-ACooP, in 1 H NMR of FNA-N-ACooP, all cysteine protons were more upfield (in ppm): 2.75 and 2.81 (Cysβ), 3.31 (CysSH), 4.39 (Cysα) and 8.17 (CysNH) and Alanine-1 protons downfield (not Ala1β): 1.35 (Ala1β), 4.54 (Ala1α) and 8.84 (Ala1NH) for Ala1.Notably, Ala1 had only one NH proton located downfield from that of free NH 3 + of unconjugated ACooP (8.04 ppm).Ala1NH and Ala1α protons were coupled to the carbonyl carbon of FNA (163.76 ppm) in the HMBC spectrum (Additional file 1: Fig. S15).Based on these data, the structure of compound FNA-N-ACooP was indeed N-acylated peptide.

Radiosynthesis and identification of [ 18 F]FNA-S-ACooP
To prepare [ 18 F]1, we decided to test the on-resin 18 F-fluorination method.Accordingly, [ 18 F]fluoride was produced by an 18 O(p,n) 18 F nuclear reaction using a cyclotron and extracted onto a PS-HCO 3 − anion-exchange cartridge (Fig. 2).The cartridge was dried by passing through dry CH 3 CN followed by nitrogen gas.A mixture of precursor 4 and DABCO in CH 3 CN/tert-BuOH was pushed through the cartridge, and we indeed observed [ 18 F]1 as the only radioactive product.In this method, it was essential to pass the precursor solution slowly so that the precursor had more time to react with the [ 18 F] fluoride bound on the cartridge.The 18 F-fluorination efficiency had a positive correlation with the amount of precursor 4 (Additional file 1: Table S1).With 8 mg and 12 mg of compound 4, 45% and 47% of [ 18 F]1 was formed in the presence of DABCO.When the amount of 4 was decreased to 2 mg and 4 mg, 9% and 23% of [ 18 F]1 was formed, respectively.Subsequently, [ 18 F]1 was purified with HPLC equipped with a semi-preparative C18 column and extracted onto two connected tC18 cartridges.[ 18 F]1 was eluted from the tC18 cartridges with CH 3 CN and ACooP peptide in borate buffer was added.
The decay-corrected radiochemical yield was 29.9% ± 2.3 (n = 4) and radiochemical purity was 96.6% ± 2.3.The molar activity was 36.2 ± 22.0 MBq/nmol at the end of synthesis.The total synthesis time was 181.5 ± 6.9 min from the end of bombardment.The stability of [ 18 F]FNA-S-ACooP in the injectable formulation (PBS with 14 mM ascorbic acid) was at least 4 h at r.t. and a longer shelf-life was not measured.LogD 7.4 of [ 18 F] FNA-S-ACooP was − 0.55 ± 0.01 (n = 3) indicating that it is a hydrophilic compound.The identity of [ 18 F]FNA-S-ACooP was confirmed with HPLC analysis by using both FNA-S-ACooP and FNA-N-ACooP as references (Fig. 3).

In vitro tissue binding
Fresh brain metastasis tissue samples were collected from a patient with lung cancer, and after snap freezing, the samples were cryosectioned to 20 µm and 8 µm thick sections.The 20-µm sections were incubated in a solution of [ 18 F]FNA-S-ACooP in PBS, rinsed with cold PBS and cold water, air dried briefly, and radioactivity was detected by autoradiography (Fig. 5A).Subsequently, the adjacent 8-µm sections were stained using antibodies against FABP3 and CD31 (biomarker for blood endothelial cells (Fig. 5B) and hematoxylin and eosin (H&E) (Fig. 5C).Autoradiography revealed heterogeneous and high focal [ 18 F]FNA-S-ACooP binding in brain metastasis tissue sections from a patient with lung cancer, and the radioactivity binding mainly co-localized with the anti-FABP3 positivity detected in immunofluorescence staining of adjacent tissue sections (Fig. 5).To further confirm the binding specificity, blocking experiments were performed using native ACooP peptide as a blocker, and the [ 18 F]FNA-S-ACooP binding was significantly reduced (P < 0.001, Additional file 1: Fig. S17).Radioactivity binding in the blocking experiments was 0.62 ± 0.09 PSL-background/mm 2 (n = 4), which was 7.5fold lower compared to the total binding of 4.64 ± 0.82 PSL-background/mm 2 (n = 4) in the unblocked experiments.

In vitro stability in rat plasma
The HPLC analysis showed that the percentage of intact [ 18 F]FNA-S-ACooP remaining after incubating in the rat plasma in vitro for 5 min, 10 min, and 15 min was 8.2% ± 2.1, 2.9% ± 0.7, and 1.6% ± 0.5 (n = 3), respectively (Additional file 1: Fig. S18).In all the samples, [ 18 F]FNA was observed as one of the radiometabolites and its amount was below 10% of the total radioactivity in the plasma samples.The chemical identity of the rest of the radiometabolites were not studied.These results indicated that [ 18 F]FNA-S-ACooP was rapidly metabolized/degraded in rat plasma, but the thioester bond between the prosthetic group and the peptide was much more stable in comparison to peptide sequence itself (Additional file 1: Fig. S18).

Discussion
In this work, we studied the radiolabeling of peptide ACooP with the activated ester of nicotinic acid [ 18 F]1 as the prosthetic compound (Fig. 2).In the chemical structure of ACooP, there is a free amino group and thiol group at the N-terminus, which provides opportunities for radiolabeling in different ways.We expected to conjugate ACooP with [ 18 F]1 by N-acylation, in a similar manner as reported in the literature about biomolecule radiolabeling with activated esters of nicotinic acid (Basuli et al. 2016;Basuli et al. 2020;Haskali et al. 2020;Keam 2021;Zhou et al. 2019).Interestingly, we observed that [ 18 F]1 conjugates with ACooP by S-acylation instead of N-acylation in a highly chemoselective manner, affording the formation of [ 18 F]FNA-S-ACooP.To confirm the identity of the [ 18 F]FNA-S-ACooP, the corresponding non-radioactive compound FNA-S-ACooP was prepared as a reference, and detailed characterization was performed with NMR and LC-MS analyzes.In addition, the N-acylated compound FNA-N-ACooP was analyzed as a reference for comparative study.The prosthetic compound [ 18 F]1 was conveniently prepared by on-resin 18 F-fluorination at r.t.without any need for azeotropic drying of [ 18 F]fluoride.The efficiency of 18 F-fluorination was increased along with the increasing amount of input precursor compound 4 according to our tests (Additional file 1: Table S1), which was in consistence with similar radiolabeling reactions reported previously (Basuli et al. 2016).This type of 18 F-fluorination is based on aromatic nucleophilic substitution, and the reaction efficiency can be enhanced with additives including DABCO at 100-140 mM (Naumiec et al. 2017).In our experiments, the use of DABCO has increased the radiolabeling efficiency of [ 18 F]1 to a limited extent and it is not essential to use DABCO.The conjugation of [ 18 F]1 with ACooP was efficient under mild reaction conditions (Additional file 1: Fig. S16).Even at a concentration of 1 mM of ACooP, 88% of conjugation efficiency was achieved at 10 min of reaction.At higher concentrations (e.g. 3 mM and 5 mM) of ACooP, a reaction time of 5 min was sufficient.When the reaction time was prolonged to 15 min, side products started to form.For quality control, the end product was analyzed with HPLC equipped with a radioactivity detector.Unfortunately, multiple peaks were observed in analytical HPLC chromatograms in the initial quality control process.We have previously observed similar problems in the radiosynthesis tests of other radiopharmaceuticals, and radiolysis has turned out to be the reason (Li et al. 2015;Silvola et al. 2018).Therefore, we tested radiolysis prevention using ascorbic acid, propylene glycol or gentisic acid, of which ascorbic acid proved to be the most effective.In the presence of 14 mM ascorbic acid in PBS at pH 5-7, [ 18 F]FNA-S-ACooP was stable for at least 4 h in the formulation solution.
To confirm the chemical identity of the radiolabeled end product, we purchased the non-radioactive N-acylated reference as a custom synthesis from United Biosystems, USA, because at the beginning we expected that end product was N-acylated compound.Accordingly, a sample of the end product was mixed with FNA-N-ACooP for co-injection for HPLC analysis.The retention times of these two peaks were indeed close to each other.However, they appeared in the wrong order in the HPLC chromatograms, with the FNA-N-ACooP peak appearing after the radiolabeled end product peak (Fig. 3A, B).In our HPLC system, the UV detector was placed before the radioactivity detector and the UV peak should precede the radioactivity peak, i.e.FNA-N-ACooP should appear slightly before and mostly overlap with the radiolabeled end product.This prompted us to prepare a non-radioactive sample under similar reaction conditions as used for the radiosynthesis for the chemical structure identification.Accordingly, we prepared 1 with an esterification reaction of 6-fluoronicotinic acid in the presence of 4-nitrophenol.The conjugation reaction of ACooP and FNA was carried out in a mixture of borate buffer (pH 8.6) and acetonitrile (70% by volume).The product was isolated with HPLC and dried under vacuum, and identified to be the S-acylated product FNA-S-ACooP.In the HPLC analysis with a sample of the radiolabeled product spiked with FNA-S-ACooP, the peaks occurred at nearly the same retention time as expected (Fig. 3A, C).Thus, it was plausible that in the radiosynthesis, the prosthetic compound [ 18 F]1 formed a thioester [ 18 F]FNA-S-ACooP with cysteine instead of an amide with alanine at the amino terminus.The S-acylation was highly chemoselective and only trace amount of N-acylated product [ 18 F]FNA-N-ACooP was observed under the used conjugation conditions (borate buffer, pH 8.6).In the identification process of [ 18 F]FNA-S-ACooP, both FNA-S-ACooP and FNA-N-ACooP were used as references.They were characterized in detail with NMR and LC-MS analyzes (see Results).
Commercial FNA-N-ACooP was prepared by solid-phase peptide synthesis, and the amino group at the N-terminus was acylated with 6-fluoronicotinic acid while the side chain of the cysteine residue was still protected.In our radiosynthesis, [ 18 F]1 was conjugated to ACooP bearing deprotected side chains and the free thiol group at the cysteine residue was available for the acylation reaction.Unexpectedly, in the radiolabeling of